A living cell carries out a complex network of more than a thousand different reactions simultaneously, with each sequence of reactions being strictly and sensitively controlled in a number of ways so that undesirable accumulations or deficiencies of intermediates and/or end products are normally prevented from occurring. As a result of this strict and sensitive control, reactions of great mechanistic complexity and stereochemical selectivity may proceed smoothly under normal physiological conditions such as ambient pressure, moderate temperature and a pH near neutrality.
In order to appreciate the complexity and selectivity of the control of metabolic networks, it is necessary first to consider specific reaction sequences such as metabolic pathways; the relationship between each pathway and the cellular architecture; the biological importance of each metabolic pathway; and the sensitive and efficient control mechanisms regulating intracellular reaction rates. The totality of intracellular reaction rates is also known as metabolic flux.
The person skilled in the art will be aware of many microbial primary and secondary metabolites and he will have access to relevant reference collections on the subject such as the authoritative Bergeys Manual. The person skilled in the art will also have to his disposal general biochemistry textbooks comprising state of the art insights into the complex world of cellular metabolism and biochemistry.
Metabolic engineering may be perceived as a purposeful redesigning of metabolic networks generating a change in and/or a redirection of the aerobic and/or the anaerobic metabolism of a microbial cell. State of the art metabolic engineering techniques have been described by among others Cameron and Chaplen (1997) in Curr. Opin. Biotechnol., vol. 8, pages 175–180, Hahn-Hägerdal et al. (1996) in Ann. New York Acad. Sci., vol. 782, pages 286–296, Stephanopoulos (1994) in Curr. Opin. Biotechnol., vol. 5, pages 196–200. Stephanopoulos and Sinskey (1993) in Trends Biotechnol., vol. 11, pages 392–396, and Cameron and Tong (1993) in Appl. Biochem. Biotechnol., vol. 38, pages 105–140.
Some microbial cells are potentially recognisable by a single characteristic trait in the form of e.g. a metabolic end product predominantly produced under a given set of growth conditions. Accordingly, a yeast may well be initially characterised by a production of ethanol in much the same way as a lactic acid bacterial cell may be potentially identifiable by a production of lactic acid. However, the complex environment wherein microbial cell metabolites are produced evidently leads not only to the formation of a single although predominant metabolite, but rather to a complex set of metabolic intermediates and end products. Many aspects of microbial metabolism and the regulation thereof are still far from being thoroughly understood.
Cellular metabolism comprises catabolism. i.e. those processes related to a degradation of complex macromolecular substances, and anabolism, or those processes concerned primarily with the synthesis of often quite complex organic molecules. Both catabolic and anabolic pathways can be perceived to occur in several stages of complexity—one being an interconversion of polymers and complex lipids with monomeric intermediates; another an interconversion of monomeric sugars, amino acids, and lipids with relatively simple organic compounds; and yet another stage being the ultimate degradation to, or synthesis from, inorganic compounds such as CO2, H2O, and NH3.
Catabolic and anabolic metabolism can be further divided into an aerobic and an anaerobic metabolism. i.e. metabolism occurring either in the presence or absence of oxygen. Many microorganisms are capable of growing in both the presence and absence of oxygen. Some microbial cells are strictly aerobic and depend absolutely upon an oxidative form of metabolism known as respiration, i.e. the coupling of energy generation to an oxidation of nutrients by oxygen.
The conversion of glucose to pyruvate in a cell undergoing active respiration, i.e. an oxidative breakdown and generation of energy from nutrient sources by means of a reaction with oxygen, results in the formation of a coenzyme in a reduced form known as nicotinamide adenine dinucleotide, or NADH. NADH is reoxidised through the mitochondrial electron transport chain in a process that generates additional energy and results in an ultimate transfer of electrons to oxygen.
The coenzyme nicotinamide adenine dinucleotide in its oxidised form (NAD+) contains a nicotinamide ring structure that is readily reducible and thus serves as an oxidising agent. Accordingly, nicotinamide adenine dinucleotide may consist in either a reduced form, NADH, or an oxidised form, NAD+. Many dehydrogenase enzymes, such as alcohol dehydrogenases, have a strong affinity for the oxidised form, NAD+. After oxidation of a substrate, the reduced form of the coenzyme. NADH, leaves the enzyme and is reoxidised by available electron-acceptor systems in the cell. The NAD+ so formed can now bind to another enzyme molecule and repeat the cycle. NAD+ and NADH differ from most substrates in that they are continually recycled.
By contrast to the oxidative metabolism of the respiratory chain, many microorganisms either can or must grow in anaerobic environments while deriving their metabolic energy from processes that do not involve oxygen. Most of such anaerobically growing microbial organisms derive their energy from fermentations characterised by energy-yielding catabolic pathways such as glycolysis, wherein a conversion of glucose results in formation of products such as e.g. ethanol and CO2.
Cellular metabolism evidently requires and generates energy, and energy-yielding metabolic pathways generate many intermediates used in numerous biosynthetic pathways. Cells mostly obtain free energy released during catabolism in the form of ATP. The chemical energy stored as ATP may be converted to other forms of energy in a process known as energy transduction.
Glycolysis is a major catabolic pathway for degradation of carbohydrates in both aerobically and anaerobically growing microbial cells. The major input to glycolysis is glucose and the pathway, comprising a total of 10 different reactions, leads to the conversion of one molecule of glucose to two molecules of pyruvate, with the concomitant generation of ATP as well as the coenzyme NADH.
The sequence of reactions between glucose and pyruvate can be considered as two distinct phases, one comprising the first five reactions and constituting an energy input phase, in which sugar phosphates are synthesised at the expense of a conversion of ATP to a less energy rich molecule in the form of ADP, and one phase comprising the last five reactions and representing an energy output phase, in which a transfer of a phosphate group to ADP leads to regeneration of ATP. The glycolytic conversion of glucose to pyruvate also involves the concomitant reduction of two moles of NAD+ to its reduced equivalent NADH.
Anaerobically growing microbial cells may reduce pyruvate produced by means of glycolysis to a variety of metabolic end products such as e.g. ethanol, lactic acid, acetic acid and carbon dioxide. Ethanol production through anaerobic fermentation of a carbon source by the yeast Saccharomyces cerevisiae is one of the best known biotechnological processes and accounts for a world production of approximately 30 billion liters per year. The ethanol yield is lower than a maximum, theoretical yield due to a formation of a number of additional products affecting the ethanol yield, such as e.g. biomass, acetate, pyruvate, succinate and glycerol. A de novo synthesis of the first four components results in a net formation of NADH, while a synthesis of glycerol occurs under simultaneous NADH consumption. As ethanol is synthesised without a net formation or consumption of NADH, glycerol formation plays an important physiological role under anaerobic growth. Glycerol formation leads to a reoxidation of NADH to NAD+ and thereby substitutes the role of oxygen as an electron acceptor.
It is known that in anaerobic cultivations of Saccharomyces cerevisiae CBS8066, approximately 10% of the carbon source is directed towards the formation of glycerol (Nissen et al., 1997: Verduyn et al., 1990). A redirection of this amount of carbon towards ethanol production is clearly desirable and would presuppose a reduction in the net formation of NADH in the synthesis of biomass and organic acids.
Accordingly, for the glycolytic pathway to operate anaerobically, i.e. in the absence of oxygen. NADH must be reoxidised to NAD+ by means of a transfer of electrons to a suitable electron acceptor so that a steady metabolic flux can be maintained. Microbial cells growing anaerobically have different ways of transferring such electrons. A simple route used by lactic acid bacteria consists of simply using NADH to reduce pyruvate to lactate, via the enzyme lactate dehydrogenase. NADH is reoxidised in the process:Pyruvate+NADHLactate+NAD−The lactic acid fermentation. i.e. conversion of glucose to lactic acid, is important in the manufacture of cheese. Another important fermentation involves a conversion of pyruvate to acetaldehyde and CO2 and a reduction of acetaldehyde to ethanol by alcohol dehydrogenase:Acetaldehyde+NADHEthanol+NAD+When carried out by yeast cells, this fermentation generates the alcohol in alcoholic beverages. Yeast cells used in baking also carry out this form of fermentation and the CO, produced by pyruvate decarboxylation causes bread to rise while the ethanol produced evaporates during baking. Among many other useful fermentations are those leading to e.g. acetic acid in the manufacture of vinegar and propionic acid in the manufacture of Swiss cheese.
Glycerol formation in cellular metabolism has at least two physiologically important roles in Saccharomyces cerevisiae—it is involved in NADH reoxidation and it acts as an efficient osmolyt that protects the cell against lysis under stress conditions.
Synthesis of biomass and organic acids, i.e. succinic acid, acetic acid and pyruvac acid, results in a net formation of intracellular NADH (Oura, 1977; van Dijken & Scheffers, 1986; Nissen et al. 1997). This has to be balanced by a mechanism in which NADH is reoxidised to NAD+ in order to avoid depletion of the NADH pool. Under anaerobic conditions. NADH reoxidation is not possible by means of the respiratory chain, which is not functioning under such conditions. Instead. NADH is reoxidised to NAD+ via formation of glycerol, since synthesis of one molecule of glycerol from glucose leads to reoxidation of one molecule of NADH.
Glycerol is also formed and accumulated inside the cell during growth under osmotic stress conditions and acts as an efficient osmolyt that protects the cell against lysis (Ansell et al., 1997; Larsson et al. (1993)). The formation of glycerol occurs via a two step reaction from dihydroxyacetone phosphate (DHAP) that is catalysed by glycerol 3-phosphate dehydrogenase and glycerol 3-phosphate phosphatase, respectively.DHAP+NADH→Glycerol-3-Phosphate+NAD+Glycerol-3-Phosphate→Glycerol+PhosphateIn order to be able to produce any metabolic product, the microbial cell needs an input in the form of both energy and readily assimilable nutrient sources. The metabolism of a microbial cell very much determines the capability of said cell to exploit nutrient sources present in an external environment. Consequently, the metabolism of a microbial cell is dynamic and the sensitive regulation, direction and redirection of said metabolism is indicative of the responses of said cell to changing environmental conditions.
Assimilable nutrients such as various sources of nitrogen, carbon, sulphur and phosphor exist in many different forms. Some of these forms may be readily assimilated by a microbial cell while others cannot be assimilated. In the case of nitrogen, it is essential that a microbial cell is capable of assimilating this nutrient source, as nitrogen forms part of among others i) amino acids in proteins, ii) nucleotides in DNA and RNA, iii) amino sugars in complex polysaccharides, and iv) heterocyclic compounds in various coenzymes.
As described above, catabolic and anabolic pathways occur in different stages of complexity and one of said stages involves the ultimate degradation to, or synthesis from, inorganic compounds such as CO2, H2O, and NH3. The majority if not all microbial cells are capable of assimilating ammonia and converting this source of nitrogen into organic nitrogen compounds—i.e. any organic compound comprising a C—N bond. Ammonia is thus a central metabolite and actually serves as a substrate for no less than five different enzymes that convert it into various organic nitrogen-comprising compounds. At physiological pH, the dominant ionic species is an ammonium ion, but all of said five reactions involve the unshared electron pair of NH3, which is therefore generally considered the reactive species.
Accordingly, microbial cells assimilate ammonia via reactions leading to the formation of either glutamate, glutamine, asparagine, or carbamoyl phosphate. Because carbamoyl phosphate is used only in the biosynthesis of arginine, urea, and the pyrimidine nucleotides, most of the nitrogen ending up in amino acids and other nitrogen comprising organic compounds is assimilated via the two amino acids glutamate and glutamine. The enzymes responsible for ammonia assimilation in a microbial cell are briefly introduced herein below.
Glutamate dehydrogenase catalyses the reductive amination of 2-oxoglutarate:2-Oxoglutarate+NH3+NAD(P)HGlutamate+NAD(P)Microbial cells growing with ammonia as their sole nitrogen source use the above reaction as a primary route for nitrogen assimilation.
Most microbial cells contain an NADPH-specific form of the glutamate dehydrogenase enzyme, as indicated above, which acts primarily in the direction of glutamate formation. Interestingly, yeast contain both a NADH-specific form and a NADPH-specific form of the enzyme, each form being appropriately regulated, with one form, NADPH, primarily involved in nitrogen assimilation and the other, NADH, functioning primarily in catabolic metabolism.
The major source of electrons for reductive biosynthesis is NADPH, nicotinamide adenine dinucleotide phosphate. NADP+ and NADPH are identical to NAD+ and NADH, respectively, except that the form has an additional phosphate esterified at C-2′ on the adenylate moiety. NAD+ and NADP+ are equivalent in their thermodynamic tendency to accept electrons and they have similar standard reduction potentials. For reasons not known, nicotinamide nucleotide-linked enzymes that act in catabolic metabolism usually use the NAD+/NADH coenzyme pair, whereas those acting in anabolic pathways tend to use NADP+/NADPH.
Glutamate synthase is an enzyme functionally related to glutamate dehydrogenase and catalyses a reaction comparable to that catalysed by glutamate dehydrogenase. However, glutamate synthase functions primarily in glutamate biosynthesis:2-Oxoglutarate+glutamine+NADPH→2 glutamate+NADP+When formed by the action of glutamate dehydrogenase, glutamate can accept a second ammonia moiety to form glutamine in a reaction catalysed by the enzyme glutamine synthetase:Glutamate+NH3+ATP→Glutamine+ADP+Pi
This enzyme is named a synthetase, rather than a synthase, because the reaction couples bond formation with the energy released from ATP hydrolysis. However, both enzymes are classified as ligases, but a synthase enzyme does not require ATP.
Glutamine synthetase of E. coli is a dodecamer, whose 12 identical subunits form two facing hexagonal arrays. The holoenzyme has a molecular weight of about 600.000. Each catalytic site is formed at an interface between polypeptide subunits within a hexamer and is made up of residues from two adjacent subunits.
Glutamine occupies a central role in the nitrogen metabolism of any microbial cell. The amide nitrogen is used in biosynthesis of several amino acids, including glutamate, tryptophan, and histidine, purine and pyrimidine nucleotides, and amino sugars. As revealed primarily based on studies in E. coli, several remarkable and quite extraordinary control mechanisms for glutamine synthetase mediated reactions interact with one another in very complex ways. The activity of glutamine synthetase is controlled by two distinct but mutually related mechanisms: Alosteric regulation by cumulative feedback inhibition and covalent modification of the enzyme mediated by a regulatory cascade.
Cumulative feedback inhibition involves the action of no less than eight specific feedback inhibitors. Those eight inhibitors are either metabolic end products of glutamine metabolism (tryptophan, histidine, glucosamine-6-phosphate, carbamoyl phosphate, CTP, and AMP), or they are indicators in various ways of the general status of amino acid metabolism (alanine, glycine). Quite remarkably, each 50,000-dalton subunit of glutamine synthetase contains binding sites for each of the eight inhibitors, as well as binding sites for substrates and products.
Each of the eight compounds alone gives only partial inhibition, but in combination the degree of inhibition is increased until a mixture of all eight provides a virtually complete blockage. This ensures that an accumulation of an end product of one pathway does not shut off the supply of glutamine needed for another pathway. Glutamine synthetase is also regulated by means of adenylylation. An enzyme molecule with all 12 sites adenylylated is completely inactive, whereas partial adenylylation yields a correspondingly partial inactivation.
Adenylylation and deadenylylation of glutamine synthetase involve a complex series of regulatory cascades. These regulatory cascades provide a responsive mechanism ensuring that, when the supply of activated nitrogen in the form of glutamine is sufficiently high, its further biosynthesis is shut down. In contrast, when activated nitrogen in the form of glutamine is low, 2-oxoglutarate accumulates and, provided that ATP is also abundant, stimulates the activity of glutamine synthetase by the converse mechanism.
An enzyme comparable to glutamine synthetase, asparagine synthetase, accounts for a significantly smaller amount of ammonia assimilation. Asparagine synthetase uses ammonia or glutamine in catalysing the conversion of aspartate to asparagine. The enzyme cleaves ATP differently from the way ATP is cleaved by glutamine synthetase. Asparagine synthetase also differs from glutamine synthetase in that glutamine is strongly preferred as a substrate over ammonia.
Carbamoyl phosphate synthetase is another enzyme involved in the assimilation of ammonia in microbial cells. Ammonia or glutamine may both serve as the nitrogen donor.NH3+HCO3−+2 ATP→carbamoyl phosphate+2ADP+Pi Glutamine+HCO3−+2ATP+H2O→Carbamoyl phosphate+2ADP+Pi+glutamate
The bacterial enzyme catalyses both reactions, although glutamine is a preferred substrate. Eukaryotic microbial cells contain two forms of the enzyme. Form I is located in the mitochondria and has a preference for ammonia as substrate, whereas form II is present in the cytosol and has a strong preference for glutamine.
Several examples of metabolically engineered microorganisms are described in the patent literature. EP 0 733 712 A1 discloses a method of production of preferably an amino acid by culturing a metabolically engineered microbial cell, preferably an Escherichia coli cell, with a supposedly increased expression or productivity of NADPH and isolating said amino acid.
WO 96/41888 discloses a yeast cell having a modified alcohol sugar fermentation due to an altered expression of a gene encoding a NADH dependent glycerol-3-phosphate dehydrogenase activity.
EP 0 785 275 A2 discloses a yeast comprising constitutive expression of a gene encoding an enzyme activity involved in hexose transport.
EP 0 645 094 A1 discloses the use of a yeast comprising a glycolytic pathway comprising a futile cycle generated by means of a constitutive expression of genes encoding fructose-1,6-biphosphatase and phosphoenolpyruvate carboxykinase.
U.S. Pat. No. 5,545,556 discloses a yeast strain having a reduced or increased production of glycerol mediated by mutations in various gene-encoded products.
None of the above disclose a microbial cell wherein the expression of a number of expressible enzyme activities involved in nutrient assimilation are either increased or decreased or eliminated in order to alter the rate of production and/or the yield of a cellular metabolite such as an intermediate product or an end product of a metabolic pathway.